The Antimicrobial Peptide γ-Thionin from Habanero Chile (Capsicum chinense) Induces Caspase-Independent Apoptosis on Human K562 Chronic Myeloid Leukemia Cells and Regulates Epigenetic Marks

Cancer is a relevant health problem worldwide. In 2020, leukemias represented the 13th most commonly reported cancer cases worldwide but the 10th most likely to cause deaths. There has been a progressive increase in the efficacy of treatments for leukemias; however, these still generate important side effects, so it is imperative to search for new alternatives. Defensins are a group of antimicrobial peptides with activity against cancer cells. However, the cytotoxic mechanism of these peptides has been described mainly for animal defensins. This study shows that defensin γ-thionin (Capsicum chinense) is cytotoxic to the K562 leukemia cells with an IC50 = 290 μg/mL (50.26 μM) but not for human peripheral blood mononuclear cells. Results showed that γ-thionin did not affect the membrane potential; however, the peptide modified the mitochondrial membrane potential (ΔΨm) and the intracellular calcium release. In addition, γ-thionin induced apoptosis in K562 cells, but the activation of caspases 8 and 9 was not detected. Moreover, the activation of calpains was detected at one hour of treatment, suggesting that γ-thionin activates the caspase-independent apoptosis. Furthermore, the γ-thionin induced epigenetic modifications on histone 3 in K562 cells, increased global acetylation (~2-fold), and specific acetylation marks at lysine 9 (H3K9Ac) (~1.5-fold). In addition, γ-thionin increased the lysine 9 methylation (H3K9me) and dimethylation marks (H3K9me2) (~2-fold), as well as the trimethylation mark (H3K9me3) (~2-fold). To our knowledge, this is the first report of a defensin that triggers caspase-independent apoptosis in cancer cells via calpains and regulating chromatin remodelation, a novel property for a plant defensin.

The plant defensins (PDs) comprise a group of PAP whose structure is composed of helix-α and β-sheet, resembling the animal and insect defensins [10,11]. The PDs are present in seeds, leaves, tubers, fruits, roots, and barks [12]. They have antibacterial, antifungal,  (10,25,50,100,200, and 300 μg/mL), and viability was evaluated by MTT assay at 24 h. Cell viability is shown as the percentage of viable cells concerning cells treated with vehicle (DMSO 1.2%). Actinomycin D (Act-D, 80 μg/mL) was used as a positive control of death. Data represent the mean of three independent experiments performed in triplicate. * Indicates statistically significant differences concerning vehicle (p < 0.05). (B) The half maximal inhibitory concentration (IC50) of γthionin on K562 cells was calculated by regression analysis; IC50 = 290 μg/mL. (C) K562 cell morphology after different treatments. Representative photographs taken by light field microscopy are shown. Scale bars: 10 μm. (D) Effect of γ-thionin on the viability of human peripheral blood mononuclear cells. Cells were treated with γ-thionin (100, 200, and 300 μg/mL), vehicle (DMSO 1.2%), and triton 1% as a positive control of death. Viability was evaluated by MTT assays at 24 h. Data represent the average of three independent experiments performed in triplicate. Data were analyzed by t-student concerning to vehicle. * Indicates statistically significant differences concerning vehicle (p < 0.05).

γ-Thionin Defensin Does Not Affect the Membrane Integrity of K562 Cells
To determine if the cytotoxicity of γ-thionin on K562 cells was related to cell membrane damage, we evaluated the plasma membrane potential using 3,3′dipropylthiadicarbocyanine iodide (DiSC3 (5)). According to the results, γ-thionin did not affect membrane electrical potential compared with the positive control (DMSO 5%) ( Cells were treated with γ-thionin (100, 200, and 300 µg/mL), vehicle (DMSO 1.2%), and triton 1% as a positive control of death. Viability was evaluated by MTT assays at 24 h. Data represent the average of three independent experiments performed in triplicate. Data were analyzed by t-student concerning to vehicle. * Indicates statistically significant differences concerning vehicle (p < 0.05).

γ-Thionin Defensin Does Not Affect the Membrane Integrity of K562 Cells
To determine if the cytotoxicity of γ-thionin on K562 cells was related to cell membrane damage, we evaluated the plasma membrane potential using 3,3 -dipropylthiadicarbocyanine iodide (DiSC3 (5)). According to the results, γ-thionin did not affect membrane electrical potential compared with the positive control (DMSO 5%) ( Figure 2).

γ-Thionin Defensin Induces Apoptosis without Activation of Caspases in K562 Cells but Induces Calpain Activation
Different reports for AMPs have described apoptosis as the primary mechanism of activated cell death [15,27]. Therefore, we evaluated the apoptosis rate at 12 and 24 h. γ-Thionin induced early apoptosis since 12 h (>30%) in K562 cells ( Figure 3A,B), which increased at 24 h (>45%) ( Figure 3A,B); this effect was similar to that shown by actinomycin D (Figure 3) used as a positive control for apoptosis. In addition, we did not detect necrosis at times evaluated.
To identify the specific apoptotic pathway activated, we evaluated the activation of caspases 8 and 9. However, we did not detect activation of both caspases at 12 and 24 h (Figure 4), which were activated by actinomycin D. Moreover, γ-thionin modified the mitochondrial membrane potential ΔΨm since 6 h ( Figure 5), reaching the level maximum at 12 h. Interestingly, γ-thionin induces intracellular calcium efflux at a similar level to PMA ( Figure 6). Altogether, results suggest that γ-thionin activates a mechanism of caspase-independent apoptosis. . γ-thionin defensin does not affect the membrane of K562 cells. Changes in the membrane potential of K562 cells were measured using a membrane potential-sensitive dye. Cells were incubated with 200 µM DiSC3 (5) for 30 min at 37 • C and then treated with γ-thionin IC 50 , vehicle, and DMSO (5%, positive control). The assay was monitored for about 3.5 h with measurements every 2 min. Arrow indicates the time at which the treatments were added. γ-thionin = IC 50 (290 µg/mL); vehicle = DMSO 1.2%.

γ-Thionin Defensin Induces Apoptosis without Activation of Caspases in K562 Cells but Induces Calpain Activation
Different reports for AMPs have described apoptosis as the primary mechanism of activated cell death [15,27]. Therefore, we evaluated the apoptosis rate at 12 and 24 h. γ-Thionin induced early apoptosis since 12 h (>30%) in K562 cells ( Figure 3A,B), which increased at 24 h (>45%) ( Figure 3A,B); this effect was similar to that shown by actinomycin D (Figure 3) used as a positive control for apoptosis. In addition, we did not detect necrosis at times evaluated.
To identify the specific apoptotic pathway activated, we evaluated the activation of caspases 8 and 9. However, we did not detect activation of both caspases at 12 and 24 h (Figure 4), which were activated by actinomycin D. Moreover, γ-thionin modified the mitochondrial membrane potential ∆Ψm since 6 h ( Figure 5), reaching the level maximum at 12 h. Interestingly, γ-thionin induces intracellular calcium efflux at a similar level to PMA ( Figure 6). Altogether, results suggest that γ-thionin activates a mechanism of caspase-independent apoptosis. Molecules 2023, 28, x FOR PEER REVIEW 5 of 16          As the above results show, we analyzed whether γ-thionin induces calpain activity at different times. Results showed that this defensin favors the activation of calpains at 2 h (~25%) concerning the vehicle, which suggests that the cytotoxicity of this defensin on K562 cells could be related to apoptosis mediated by these molecules (Figure 7). In support of the above, the block of calpain activity with the specific inhibitor N-Acetyl-Leu-Leunorleucinal decreased the apoptosis induced by γ-thionin in K562 cells (~19%) (Figure 8). Measurements were performed for 5 min. For readings, a baseline fluorescence of 1 min was established, then the treatments were placed, and the fluorescence intensity was monitored for another 5 min. The panel shows representative plots (A) and relative fluorescence intensities for intracellular calcium release (B). Arrow indicates the time at which the treatments were added. PMA (3 µM) was used as a positive control; γ-thionin = IC 50 (290 µg/mL); vehicle = DMSO 1.2%.
As the above results show, we analyzed whether γ-thionin induces calpain activity at different times. Results showed that this defensin favors the activation of calpains at 2 h (~25%) concerning the vehicle, which suggests that the cytotoxicity of this defensin on K562 cells could be related to apoptosis mediated by these molecules (Figure 7). In support of the above, the block of calpain activity with the specific inhibitor N-Acetyl-Leu-Leu-norleucinal decreased the apoptosis induced by γ-thionin in K562 cells (~19%) (Figure 8).

Discussion
In developing alternative therapies against cancer, plant antimicrobial peptides have attracted attention for their cytotoxicity, efficacy, and selectivity against some types of cancer [2,3]. Hence, we showed that the plant defensin γ-thionin (Capsicum chinense) is cytotoxic against K562 leukemia cells and that its mechanism of action is by caspaseindependent apoptosis mediated by calpains. In addition, γ-thionin regulates histone epigenetic marks.
γ-thionin was cytotoxic to K562 cells in a concentration-dependent manner, with an IC50 = 290 μg/mL (50.26 μM) (Figure 1). Interestingly, the γ-thionin IC50 was higher concerning the effect of other plant defensins on leukemia cells, such as NAD1 (2.4 μM), coccinin (30 μM), phaseococcin (40 μM) and gymnin (50 μM) [28][29][30]. In addition, γthionin defensin was not cytotoxic against human peripheral blood mononuclear cells, as previously reported [26], suggesting that it could be selective against cancer cells; however, more experiments are needed to prove it. In addition, it would be important to evaluate whether γ-thionin defensin can act synergistically with cytostatic agents, e.g., doxorubicin, as reported for avocado defensin PaDef [24]. This could reduce the side effects of conventional treatments and expand the therapeutic possibilities of AMPs. Finally, it would be interesting to analyze the possibility to target cancer cells using this peptide in combination with nanoparticles.
The primary mechanism of cytotoxicity reported for plant defensins on leukemia cells involves membrane damage; this is the case for defensin NaD1 (N. alata) and TPP3 (L. esculentum) [13,14]. However, γ-thionin did not affect the membrane integrity of K562 cells (Figure 2), which suggests a different mechanism for this peptide. Notably, the cell morphology was affected, showing structures like-apoptotic bodies ( Figure 1D); an observation that was corroborated by flow cytometry (Figure 3).
Searching for molecules that activate apoptosis in cancer cells and whose effects are related to modifications in chromatin remodeling could be an attractive anticancer strategy. Apoptosis is a process of cell death that occurs through two principal mechanisms, the intrinsic and extrinsic pathways, and is a cytotoxic mechanism described

Discussion
In developing alternative therapies against cancer, plant antimicrobial peptides have attracted attention for their cytotoxicity, efficacy, and selectivity against some types of cancer [2,3]. Hence, we showed that the plant defensin γ-thionin (Capsicum chinense) is cytotoxic against K562 leukemia cells and that its mechanism of action is by caspaseindependent apoptosis mediated by calpains. In addition, γ-thionin regulates histone epigenetic marks.
γ-thionin was cytotoxic to K562 cells in a concentration-dependent manner, with an IC 50 = 290 µg/mL (50.26 µM) (Figure 1). Interestingly, the γ-thionin IC 50 was higher concerning the effect of other plant defensins on leukemia cells, such as NAD1 (2.4 µM), coccinin (30 µM), phaseococcin (40 µM) and gymnin (50 µM) [28][29][30]. In addition, γ-thionin defensin was not cytotoxic against human peripheral blood mononuclear cells, as previously reported [26], suggesting that it could be selective against cancer cells; however, more experiments are needed to prove it. In addition, it would be important to evaluate whether γ-thionin defensin can act synergistically with cytostatic agents, e.g., doxorubicin, as reported for avocado defensin PaDef [24]. This could reduce the side effects of conventional treatments and expand the therapeutic possibilities of AMPs. Finally, it would be interesting to analyze the possibility to target cancer cells using this peptide in combination with nanoparticles.
The primary mechanism of cytotoxicity reported for plant defensins on leukemia cells involves membrane damage; this is the case for defensin NaD1 (N. alata) and TPP3 (L. esculentum) [13,14]. However, γ-thionin did not affect the membrane integrity of K562 cells (Figure 2), which suggests a different mechanism for this peptide. Notably, the cell morphology was affected, showing structures like-apoptotic bodies ( Figure 1D); an observation that was corroborated by flow cytometry (Figure 3).
Searching for molecules that activate apoptosis in cancer cells and whose effects are related to modifications in chromatin remodeling could be an attractive anticancer strategy. Apoptosis is a process of cell death that occurs through two principal mechanisms, the intrinsic and extrinsic pathways, and is a cytotoxic mechanism described for plant defensins. For example, radish RsAFP2 and Heuchera sanguinea HsAFP1 defensins induce apoptosis in Candida albicans [31,32]. In concordance, we demonstrated that avocado PaDef defensin activates caspase-dependent apoptosis in MCF-7 and K562 cells [15,16]. Interestingly, this study provides evidence that γ-thionin cytotoxicity on K562 cells occurs through caspase-independent apoptosis because the activity of caspases 8 and 9 was not detected (Figure 4). In agreement, the mitochondrial membrane potential (∆Ψm) and the intracellular calcium release of K562 cells were modified by γ-thionin at short intervals ( Figures 5 and 6); both phenomena are characteristic of caspase-independent apoptosis mediated by calpains [18][19][20][21]. Accordingly, the activity of calpains was detected after 1 h of treatment (Figure 7). Similarly, the human cathelicidin LL-37 activates calpain-dependent apoptosis in Jurkat T leukemia cells [22]. In addition, the antimicrobial peptide epinecidin-1 (epi-1) from the orange-spotted grouper fish (Epinephelus coioides) activates caspaseindependent cell death in synovial sarcoma cells SW982 cells, increased intracellular calcium levels, ROS production, and calpain activity [33]. However, both AMPs are not defensins, LL-37 is a cathelicidin, and epi-1 is an α-helix peptide. This is the first report of defensininducing caspase-independent apoptosis mediated by calpains in leukemia cells.
Epigenetic alterations in histones are essential in developing and maintaining diseases such as cancer. The recovery of altered cellular phenotypes through epigenetic modifications is possible because, unlike genetic determinants, epigenetic alterations are reversible, which makes the search for molecules with epigenetic activity in cancer attractive as a treatment alternative for these diseases. For this reason, we analyzed whether the concentration at which γ-thionin triggers K562 cell death induces epigenetic modifications, as reported for PaDef defensin in the Jurkat leukemia cell line [24]. The results indicate that γ-thionin increased acetylation and methylation marks on histone 3 in K562 cells (Figures 9 and 10). The increase in acetylation marks could result from inhibiting histone deacetylases (HDACs). This agrees with a previous report showing that γ-thionin inhibits the HDACs activity in MCF-7 breast cancer cells and whose inhibition increases the acetylation mark of lysine 9 on histone 3 (H3K9Ac) [34]. In addition, the inhibitory HDACs activity of γ-thionin could explain why calpain blockade decreased apoptotic activity ( Figure 8) but did not completely block it. Several mechanisms by which inhibitors of HDACs (iHDACs) trigger apoptosis have been described [35]. Some are caspaseindependent; for example, suberoylanilide hydroxamic acid (SAHA) induces apoptosis through a mitochondrial pathway in which caspases are not involved [36]. However, further experiments to evaluate whether γ-thionin affects the HDAC activity in K562 cells are necessary to clarify this point. Regarding the increase in methylation marks, it is necessary to evaluate the activity of either histone methyl transferases or histone methyl transferase inhibitors, which could give us a broader picture of the γ-thionin effects on K562 cells. On the other hand, it has been documented that iHDACs can inhibit demethylase enzymes, such is the case of the iHDACs AR42 and MS-275 that transcriptionally repress some members of the JARID1 family, in charge of the demethylation of histone 3 at lysine 4 (H3K4me) with a consequent increase in the methylation mark [36]. This could give a guideline on the duality of γ-thionin modified marks; however, as mentioned above, evaluating γ-thionin on HDAC activity is necessary. In conclusion, the results showed in this work indicate that γ-thionin activates caspase-independent apoptosis via calpains in K562 cells and regulates epigenetic marks.

Mammalian Cell Culture
The human leukemia cell line K562 was obtained from American Type Culture Collection. Cells were routinely cultured in RPMI-1640 Media (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% (v/v) fetal bovine serum (Corning, NY, USA) and 100 U/mL of penicillin and streptomycin (Gibco, Waltham, MA, USA) and grown in an atmosphere of 5% CO 2 at 37 • C, as reported [16]. In addition, Human Peripheral Blood Mononuclear Cells (PBMC) were obtained from the blood of healthy men volunteers. The PBMC were isolated by density gradient centrifugation using Ficoll-Paque Tm Plus (GE Healthcare, Chicago, IL, USA), and the cells were cultured under the same conditions mentioned above.

Cell Viability Assays
The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide] assay was used to assess the cytotoxicity of γ-thionin [16]. Briefly, K562 cells were synchronized in the RPMI-1640 medium without serum (12 h). Cells were seeded in a 96-well plate at a density of 2 × 10 4 cells/well and cultured with γ-thionin peptide at various concentrations (10,25,50,100,200, and 300 µg/mL). After 24 h of incubation, 10 µL of MTT solution (5 mg/mL, Sigma-Aldrich, St. Louis, MO, USA) was added to each well, and plates were incubated at 37 • C in 5% CO 2 for 4 h. Then, 100 µL of isopropyl alcohol: HCl (19:1) was added to dissolve formazan crystals and was incubated by 20 min. Absorbance was measured at 595 nm using a microplate reader (iMark Microplate Absorbance Reader, BioRad, CA, USA). Actinomycin D was used as cell death-positive control. The results were reported as the percentage of viability concerning vehicle (DMSO 1.2%). Using Excel (Microsoft), the half maximal inhibitory concentration (IC 50 = 290 µg/mL) was determined by regression analysis. Additionally, SYTO ®® 9 green-fluorescent nucleic acid stain and propidium iodide was used to validate IC 50 by flow cytometry, according to the manufacturer's instructions. K562 cells were prepared as previously described and treated for 24 h with IC 50 . The measurement was carried out using a BD Accuri™ C6 flow cytometer (BD Biosciences, San Jose, CA, USA). IC50 concentration of γ-thionin was used for the rest of the experiments.

Apoptosis and Caspases Activation
The apoptosis rate was assessed using a BD Accuri™ C6 flow cytometer (BD Biosciences, San Jose, CA, USA) employing Annexin V (Annexin V, Alexa Fluor 488 conjugate, Invitrogen, Carlsbad, CA, USA) and 7AAD (Bio Legend, San Diego, CA, USA) according to the manufacturer's instructions [15]. A total of 10,000 events were collected. The data were analyzed using FlowJo software version 10.4 (TreeStar Inc., Ashland, OR, USA). Actinomycin D (Sigma-Aldrich, 80 µg/mL) was used as a positive control.
The activation of caspases 8 and 9 was evaluated with Caspase-Glo 8 and 9 kits (Promega, Madison, WI, USA) according to the manufacturer's instructions [16]. K562 cells (6 × 10 4 / well) were seeded in white 96-well plates and incubated with IC 50 or vehicle in the serum-free medium for 12 or 24 h. The luminescence was detected using a Varioskan spectrophotometer (Thermo Scientific). Actinomycin D (Sigma-Aldrich, 80 µg/mL) was used as a positive control.

Evaluation of Mitochondrial Membrane Potential (∆Ψm)
The changes in mitochondrial membrane potential were monitored using the JC-1 dye (BD Biosciences) in a BD Accuri™ C6 flow cytometer (BD Biosciences) [15]. The K562 cells (1 × 10 5 cells/well) were cultured in 96-well plates and treated with γ-thionin IC 50 or vehicle for 6, 12, and 24 h. The cells were treated according to the manufacturer's instructions. The fluorescence was measured in a BD Accuri ™ C6 flow cytometer (BD Biosciences). The data were analyzed using FlowJo software version 10.4 (TreeStar, Inc., San Carlos, CA, USA). Actinomycin D (Sigma-Aldrich, 80 µg/mL) was used as a positive control.

Calcium Efflux Testing
Calcium efflux was measured by flow cytometry in a BD Accuri™ C6 flow cytometer (BD Biosciences) using a Calcium Assay Kit (BD Biosciences) according to the manufacturer's instructions [15]. The K562 cells (1 × 10 5 cells/ well) seeded in 96-well plates were incubated with the indicator dye for 1 h. A baseline fluorescence (3 min) was established and treatments were added (γ-thionin IC 50 or vehicle). The fluorescence intensity was monitored by flow cytometry without interruption for another 3 min. The data were analyzed with Excel (Microsoft). The Phorbol Myristate Acetate (3 µM; PMA, Sigma-Aldrich) was used as a positive control.

Activation of Calpains
The activation of calpains was assessed with the Calpain-Glo kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. Briefly, K562 cells (6 × 10 4 / well) were seeded in white 96-well plates and incubated with IC 50 or vehicle in serum-free medium for 1, 2, 4, 12, and 24 h. The luminescence data were collected in a Varioskan spectrophotometer (Thermo Scientific). K562 cells were incubated with the calpains inhibitor N-Acetyl-Leu-Leu-norleucinal (5 mg/mL) for 1 h and then treated with γ-thionin IC 50 by 24 h, and apoptosis rate was determined as described above. The cisplatin (1 µg/mL, Pisa) was used as a positive control.
For western blot analysis, the histones were separated in a 15% SDS-PAGE gel and transferred to a PVDF membrane by semi-dry transfer, as reported [37]. The membranes were blocked with 5% nonfat dry milk in cold PBS at 4 • C overnight. Furthermore, the membranes were washed three times with cold TBS, and the primary antibody (1:1000) was added and incubated at 4 • C overnight. The following antibodies were used to evaluate: global acetylation (H3K9, K14, K18, K23, K27) (Abcam ab47915, Boston, MA, USA), H3K9ac (Abcam, ab10812) for acetylation, H3K9me2 (Abcam Ab1220), and H3K9me3 (Abcam, ab8898) for di-and tri-methylation, respectively. In addition, the antibody for histone H3 (Abcam, ab1791) was used for loading control. Furthermore, the membranes were incubated with horseradish peroxidase-coupled anti-IgG secondary antibody (1:3000) (Cell Signaling Technology, Danvers, MA, USA). The membranes were washed three times and revealed with Western ECL. The intensity of the signals was quantified by densitometry using the ImageJ software. Sodium butyrate (3.5 mM) was used as a positive control for H3K9 acetylation induction and negative for H3K9me2/3 methylation induction. Data were normalized concerning histone H3 and shown as the relative level of expression concerning the vehicle.

Statistical Analysis
The data were obtained from three independent experiments performed in triplicate. The significance of the differences was assessed using Student's t-test with PRISM 8.02. software. A p < 0.05 was considered significant.

Conclusions
The plant defensin γ-thionin triggers caspase-independent apoptosis via calpains in K562 cells and regulates epigenetic marks, a novel property of this plant defensin.